Retinal microglial cells are immune cells of the retina and participate in the retinal immune response. In recent years, it has been found that microglia plays an important role in the pathogenesis of diabetic retinopathy (DR), and is involved in the pathological process of neurodegeneration and microvascular disease in DR. Understanding the function of retinal microglial cells and their role in the pathogenesis DR may open up new avenues for the treatment of DR through the precise regulation of microglia
ObjectiveTo explore the effect and mechanism of directive differentiation of microglia by SN50 on hypoxia-caused neurons injury in mice.MethodsThe microglia were isolated and purified from brain tissue of new-born BALB/c mice through differential velocity adherent and vibration technique. The quantity of the microglia was identified by immunofluorescence staining of inducible nitric oxide synthetase (iNOS) and ionized calcium binding adapter molecule 1 (Iba1) and real-time fluorescence quantitative PCR (qRT-PCR) for special expression genes [iNOS, CD32, and interlenkin 10 (IL-10)]. Then the microglia were cultured with SN50, and the expressions of nuclear factor κB (NF-κB), differentiation-related genes (iNOS, CD11b, IL-10, and CD206), and apoptosis were detected by Western blot, qRT-PCR, and flow cytometry, respectively. The hypoxia model of neuron was established, and the cell apoptosis was evaluated by MTT after 0, 2, 6, 12, 24, and 48 hours of anoxic treatment. The apoptosis related markers (Bcl-2 and Caspase-3) were measured by Western blot and flow cytometry. In addition, the neurons after anoxic treatment were co-cultured with SN50 treated microglia (experimental group) and normal microglia (control group) for 24 hours. And the cell viability and apoptosis related markers (Bcl-2 and Caspase-3) were also measured.ResultsImmunofluorescence staining and qRT-PCR analysis showed that the cells expressed the specific proteins and genes of microglia. Compared with the normal microglia, the relative expressions of NF-κB protein and iNOS and CD11b mRNAs in the microglia treated with SN50 significantly decreased (P<0.05), the relative expressions of IL-10 and CD206 mRNAs significantly increased (P<0.05), and the cell apoptosis rate had no significant change (P>0.05). Compared with the normal neurons, the cell viability, the relative expressions of Bcl-2 and Caspase-3 proteins after anoxic treatment significantly decreased (P<0.05), while the relative expressions of cleaved-Caspase-3 protein and cell apoptosis rate of neurons significantly increased (P<0.05). In the co-culture system, the cell viability, the relative expressions of Bcl-2 and Caspase-3 proteins were significantly higher in experimental group than those in control group (P<0.05), while the relative expressions of cleaved-Caspase-3 protein and cell apoptosis rate were significantly lower in experimental group than those in control group (P<0.05).ConclusionSN50 can induce the microglia differentiation into M2 type through NF-κB pathway. The SN50-induced microglia can protect neurons from hypoxic injury.
ObjectiveTo investigate the effects of FTY720 on retinal photoreceptor cells and microglial following light-induced degeneration in rat retina. Methods120 Sprague-Dawley rats were randomly divided into four groups including FTY720 group, solvent control group, model group and normal group. The rats of normal group were not intervened. The FTY720 group, solvent control group and model group establish retinal light injury mode. FTY720 was injected into abdominal cavity of the rats in FTY720 group 0.5 hours before light exposure. 50% dimethylsulfoxide was injected into abdominal cavity of the rats in solvent control group. The expressions of microglial cells in rat retinal were quantified using flow cytometry, the expressions of interleukin (IL)-1βwere examined by enzyme-linked immuno sorbent assay at 6 hours, 1 day, 3 days, 7 days after light exposure. The apoptosis of retinal photoreceptor cells were measured by terminal-deoxynucleoitidyl transferase mediated nick end labeling at 1 day after light exposure. The morphological change of retinal were viewed by haematoxylin and eosin staining at 7 days after light exposure. ResultsThe expressions of microgilal and IL-1βbegan to rise at 1 day after light exposure, reached at peak at 3 days and decreased at 7 days. The expressions of IL-1βand microglial in FTY720 group were significantly lower than solvent control group and model group, but higher than normal group (P < 0.05).One day after exposure to light, the apoptosis cell ratio in normal group, model group, solvent control group and FTY720 group were 0, (87.66±2.50)%, (86.00±2.44)%, (49.66±2.80)%. The apoptosis cell in FTY720 group were higher than normal group, lower than solvent control group and model group (P < 0.05). Seven days after exposure to light, the retinal in normal group was structured and the cell was arranged well, the cell in solvent control group and model group was irregular arrangement and the outer nuclear layer (ONL) was thin after light exposure. The thickness of the ONL in FTY720 group was significantly higher than solvent control group and model group, below normal group. ConclusionFTY720 can prevents retinal photoreceptor cells from apoptosis and inhibits activation of microglial.
OBJECTIVE :To investigale effect of subretinal fluld(SRF)on proliferalion of the cellular elements of PVR. METHOD:The effect of SRF of 28 patients with rhegmatogenous retinal detachment proliferation of the cultured human retinal pigment epithelial cells(RPE),retinal glial cells (RG),and fibroblast (FB)was observed and detected by the methods of cell-counting and 3H-TdR in DNA synthesis. RESULTS:The range of proliferatinn-stimulating activity was 52.5%~233.3%, 36.4% ~ 177.8%，55.4% ~277.8% above the baseline in 1:10 dilution of these 3 kinds ,of cellular elements,and there was no significant difference among them. CONCLUSION ;The stimulating effect of SRF on the cellular proliferation was thougt to be due to the actions from certain growth factors. （Chin J Ocul Fundus Dis,1996,12: 233-235）
Objective To investigate the effect of astragaloside A (AS-A) on the photoreceptor degeneration induced by sodium iodate (NaIO3) and its related mechanism. MethodsSixty healthy male C57BL/6J mice, aged 6-8 weeks, were randomly divided into normal control (NC) group, NaIO3 group, and AS-A group, with twenty mice in each group. 30 min before modeling, AS-A group mice were intraperitoneally injected with 100 μl AS-A at a dose of 100 mg/kg body weight. 30 min later, mice in NaIO3 group and AS-A group were intraperitoneally injected with 100 μl NaIO3 at a dose of 30 mg/kg body weight. Subsequently, AS-A group mice were administered AS-A twice daily at 12 h intervals until the end of the experiment. On day 1 post-modeling, zonula occludens-1 (ZO-1) immunohistochemistry was performed to observe the structure of retinal pigment epithelium (RPE) cells; real-time quantitative polymerase chain reaction (qPCR) was conducted to detect the mRNA expression of various retinal chemokine ligand-2 (Ccl2), interleukin-1 beta (Il-1β), mixed lineage kinase domain-like protein (Mlkl), receptor-interacting protein kinase 3 (Ripk3), and tumor necrosis factor (Tnf). On day 3 post-modeling, immunohistochemistry was performed to observe the expression of ionized calcium binding adaptor molecule 1 (Iba1) and glial fibrillary acid protein (GFAP) in the retina; TdT-mediated dUTP nick-end labeling (TUNEL) assay was used to detect photoreceptor cell death in each group. On day 4 post-modeling, fundus morphology of mice in each group was observed by fundus color photography and optical coherence tomography (OCT). Hematoxylin-eosin staining (HE) was used to observe the morphological structure of the retina in each group. Inter-group comparisons between two groups were conducted using independent samples t-test, while comparisons among three groups were performed using one-way ANOVA. ResultsFundus color photography and OCT examination showed that a large number of scattered yellow-white subretinal nodular structures in the fundus of NaIO3 group mice, and a large number of strong reflection areas in the RPE layer. The number of strong reflection areas in the RPE layer was reduced in the AS-A group. Immunohistochemical analysis of ZO-1 showed that ZO-1 was largely lost on the RPE cell membrane in that NaIO3 group; whereas in the AS-A group, ZO-1 was evenly distributed on the RPE cell membrane. HE staining results showed circular black deposits were visible in the RPE layer of the NaIO3 group, and the inner and outer segments of photoreceptors were severely damaged, with a significant decrease in the number of outer nuclear layer (ONL) cell nuclei; whereas in the AS-A group, the RPE layer pigments were orderly, the inner and outer segments of photoreceptors were intact, and the number of ONL cell nuclei significantly increased. The results of TUNEL staining show that numerous TUNEL-positive cell nuclei were observed in the ONL of the retina in the NaIO3 group, while the number of TUNEL-positive cell nuclei in the ONL of the retina was significantly reduced in the AS-A group, with statistically significant differences (t=2.66, P＜0.05). The analysis of qPCR data showed that compared with the AS-A group, the relative expression levels of Mlkl, Ripk3, Ccl2, Il-1β and Tnf mRNA in the retina were significantly increased in the NaIO3 group, with statistically significant differences (F=39.18, 10.66, 53.51, 41.40, 24.13; P＜0.001). Immunohistochemical staining results showed that compared with NC group and AS-A group, the positive expression of GFAP in retina of NaIO3 group was significantly increased, and the difference was statistically significant (F=9.62, P＜0.05). ConclusionAS-A antagonizes NaIO3-induced photoreceptor degeneration in part by inhibiting photoreceptor cell death and neuroinflammation. Meanwhile, AS-A treatment protects against NaIO3-triggered perturbation of retinal homeostasis.
ObjectiveTo observe the effect of metformin on the polarization state and photoreceptor cell activity of microglia (BV2 cells) in a high glucose environment. MethodsAn experimental study. BV2 cells were divided into a control group, a high glucose group, and a metformin+high glucose group. The cells in the high glucose group were cultured with 75 mmol/L glucose in the medium; the cells in the metformin+high glucose group were pretreated with 2 mmol/L metformin for 12 h and then placed in 75 mmo/L glucose concentration medium. The relative expression of M1 marker inducible nitric oxide synthase (iNOS), CD86 and M2 markers arginase 1 (Arg-1), and CD206 protein were detected by Western blot. Interleukin (IL)-6, tumor necrosis factor (TNF)-α, IL-4 were detected by enzyme-linked immunosorbent assay (ELISA). BV2 cells were co-cultured with mouse retinal photoreceptor cells (661W cells) for 24 h. The proliferation rate of 661W cells in each group was measured by methyl thiazolyl tetrazolium (MTT) colorimetric assay; the apoptosis rate of 661W cells in each group was measured by flow cytometry and terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL). An independent sample t-test was used for comparison between groups. ResultsWestern blot assay showed that the relative expression of iNOS and CD86 protein was increased and the relative expression of Arg-1 and CD206 protein was decreased in BV2 cells in the high glucose group compared with the control group, and the differences were all statistically significant (t=－16.783, －11.605, 4.325, 4.649; P＜0.05); compared with the high glucose group, the relative expression of iNOS and CD86 protein was decreased and the relative expression of Arg-1 and CD206 protein was increased in BV2 cells in the metformin + high glucose group compared with the high glucose group, and the differences were all statistically significant (t=7.231, 5.560, －8.035, －8.824; P＜0.01). ELISA results showed that compared with the control group, the BV2 cells in the high glucose group had increased IL-6, TNF-α content and IL-4 content was decreased in BV2 cells in the high glucose group compared with the control group, and the differences were all statistically significant (t=－64.312, －127.147, 71.547; P＜0.001); compared with the high glucose group, IL-6 and TNF-α content was significantly decreased and IL-4 content was significantly increased in BV2 cells in the metformin+high glucose group, and the differences were all statistically significant (t=44.426, 83.232, －143.115; P＜0.001). After co-culture of BV2 cells with 661W cells for 24 h, the results of MTT colorimetric assay showed that compared with the control group, the activity of 661W cells in the high glucose group was significantly reduced, and the difference was statistically significant (t=7.456, P＜0.01); compared with the high glucose group, the activity of 661W cells in the metformin+high glucose group was increased (t=－3.076, P＜0.05). TUNEL method and flow cytometry showed that the apoptosis rate of 661W cells in the high glucose group was significantly higher compared with the control group, and the differences were both statistically significant (t=－22.248, －22.628; P＜0.001); compared with the high glucose group, the apoptosis rate of 661W cells in the metformin+high glucose group was significantly decreased, and the difference was statistically significant (t=11.767, 6.906; P＜0.001, 0.01). ConclusionIn the high glucose environment, metformin inhibited the inflammatory response and attenuated the apoptosis of photoreceptor cells by regulating the polarization of microglia toward the M2 type.
ObjectiveTo observe the role of Notch signaling pathway inhibitor in differentiation process of stem cells derived from retinal Müller cells into the ganglion cell. MethodsRetinas of Sprague Dawley rat at postnatal 10-20 days were dissociated from eye balls. The third passage of Müller cells was used in this experiment, which cultured by repeated incomplete pancreatic enzyme digestion method. The retinal Müller cells were induced in the serum-free dedifferentiation medium. The cell proliferation state was observed under an inverted microscope. The expression of the specific markers Nestin and Ki-67 of retinal stem cells was measured by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. The positive rate of nucleus was detected by Edu. The retinal stem cells was divided into Gamma secretase inhibtor-I (GSI) group and control group, the rate of ganglion cells was counted by using immunofluorescence staining. ResultsThe cell proliferation had gathered to form a sphere. Immunofluorescence staining showed that the expressions of Nestin and Ki-67 were (92.94±6.48%) and (85.96±6.04%) respectively. Edu positive rate of nucleus was (82.80±6.65)%. RT-PCR and Western blot further confirmed the high expression of Nestin and Ki-67 in the cell spheres but not in the Müller cells. The positive rate of ganglion cells were (16.98±2.87)% and (11.17±0.71)% in GSI group and control group respectively, with the significant difference (t=3.210, P=0.002). ConclusionNotch signaling pathway is an important regulatory gene in stem cells differentiated into retinal ganglion cell.
The human hereditary retinal degeneration is one of the main cause of irreversible blindness in the world. the mechanisms leading to retinal photoreceptor degeneration are not entirely clear. However, microglia acting as innate immune monitors are found to be activated early in retinal degeneration in many retinitis pigmentosa animal models. These activated microglia are involved in phagocyte rod cell fragments of degenerated retina, and also produce high levels of cytotoxic substances such as pro-inflammatory cytokines and chemokines, which aggravate the death of adjacent healthy photoreceptor cells. It suggests that microglia activation plays an important role in photoreceptor degeneration. At the same time, a series of studies have confirmed that some drugs can prevent or reduce neuronal death and slow the occurrence and progression of retinal degeneration by interfering with abnormal activation of microglia. It is expected to be a new choice for the treatment of hereditary retinal degeneration.
Retinal macrophages and (or) microglial cells play important roles in regulating inflammation, angiogenesis and tissue repairing, thus affect the development and prognosis of ischemic retinal disease, ocular immune diseases and ocular tumors. Reversing the polarization imbalance of these cells may provide new therapeutic strategies for ischemic retinal disease and ocular immune diseases. The duality of the polarization direction of these cells is still controversial in the inflammatory reaction and pathological angiogenesis of ischemic retinal disease. Meanwhile, the plasticity and diversity of the function need to be further studied and discussed.
Objective To investigate the effects of removing microglia from spinal cord on nerve repair and functional recovery after spinal cord injury (SCI) in mice. MethodsThirty-nine 6-week-old female C57BL/6 mice were randomly divided into control group (n=12), SCI group (n=12), and PLX3397+SCI group (n=15). The PLX3397+SCI group received continuous feeding of PLX3397, a colony-stimulating factor 1 receptor inhibitor, while the other two groups were fed a standard diet. After 14 days, both the SCI group and the PLX3397+SCI group were tested for ionized calcium binding adapter molecule 1 (Iba1) to confirm that the PLX3397+SCI group had completely depleted the spinal cord microglia. The SCI model was then prepared by clamping the spinal cord in both the SCI group and the PLX3397+SCI group, while the control group underwent laminectomy. Preoperatively and at 1, 3, 7, 14, 21, and 28 days postoperatively, the Basso Mouse Scale (BMS) was used to assess the hind limb function of mice in each group. At 28 days, a footprint test was conducted to observe the gait of the mice. After SCI, spinal cord tissue from the injury site was taken, and Iba1 immunofluorescence staining was performed at 7 days to observe the aggregation and proliferation of microglia in the spinal cord. HE staining was used to observe the formation of glial scars at the injury site at 28 days; glial fibrillary acidic protein (GFAP) immunofluorescence staining was applied to astrocytes to assess the extent of the injured area; neuronal nuclei antigen (NeuN) immunofluorescence staining was used to evaluate neuronal survival. And 5-hydroxytryptamine (5-HT) immunofluorescence staining was performed to assess axonal survival at 60 days. Results All mice survived until the end of the experiment. Immunofluorescence staining revealed that the microglia in the spinal cord of the PLX3397+SCI group decreased by more than 95% compared to the control group after 14 days of continuous feeding with PLX3397 (P<0.05). Compared to the control group, the BMS scores in the PLX3397+SCI group and the SCI group significantly decreased at different time points after SCI (P<0.05). Moreover, the PLX3397+SCI group showed a further decrease in BMS scores compared to the SCI group, and exhibited a dragging gait. The differences between the two groups were significant at 14, 21, and 28 days (P<0.05). HE staining at 28 days revealed that the SCI group had formed a well-defined and dense gliotic scar, while the PLX3397+SCI group also developed a gliotic scar, but with a more blurred and loose boundary. Immunofluorescence staining revealed that the number of microglia near the injury center at 7 days increased in the SCI group than in the control group, but the difference between groups was not significant (P>0.05). In contrast, the PLX3397+SCI group showed a significant reduction in microglia compared to both the control and SCI groups (P<0.05). At 28 days after SCI, the area of spinal cord injury in the PLX3397+SCI group was significantly larger than that in SCI group (P<0.05); the surviving neurons significantly reduced compared with the control group and SCI group (P<0.05). The axonal necrosis and retraction at 60 days after SCI were more obvious. ConclusionThe removal of microglia in the spinal cord aggravate the tissue damage after SCI and affecte the recovery of motor function in mice, suggesting that microglia played a neuroprotective role in SCI.